Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells / 北京大学学报(医学版)

ABSTRACT

Objective:To explore the effect of ubiquitin-specific protease 42(USP42)on osteogenic differentiation of human adipose-derived stem cells(hASCs)in vivo and in vitro.Methods:A combina-tion of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy.Alkaline phosphatase(ALP)staining and quantification,alizarin red S(ARS)staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group(knockdown group and overexpression group)and the control group.Quantitative re-verse transcription PCR(qRT-PCR)was used to detect the expression levels of osteogenesis related genes in the experimental group and control group,and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group.Nude mice ectopic im-plantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo.Results:The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group,and those in overexpression group were significantly higher than those in control group.After 7 days of osteogenic induction,the ALP activity in the knockdown group was sig-nificantly higher than that in the control group,and ALP activity in overexpression group was significantly lower than that in control group.After 14 days of osteogenic induction,ARS staining was significantly deeper in the knockdown group than in the control group,and significantly lighter in overexpression group than in the control group.The results of qRT-PCR showed that the mRNA expression levels of ALP,os-terix(OSX)and collagen type Ⅰ(COL Ⅰ)in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction,and those in overexpression group were signifi-cantly lower than those in control group.The results of Western blotting showed that the expression levels of runt-related transcription factor 2(RUNX2),OSX and COL Ⅰ in the knockout group were significant-ly higher than those in the control group at 14 days after osteogenic induction,while the expression levels of RUNX2,OSX and COL Ⅰ in the overexpression group were significantly lower than those in the control group.Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group.Conclusion:Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vi-vo,and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs,and USP42 can provide a potential therapeutic target for bone tissue engineering.